JP5408839B2 - Cell cycle analysis method - Google Patents

Description

  The present invention relates to a cell cycle analysis method, and more particularly to a cell cycle related data acquisition method when a cell or tissue is recognized by an image and analyzed using data obtained from the image.

When analyzing the cell cycle with an imaging cytometer, it is common to stain the cell nucleus and determine the cell cycle from its DNA content and maximum brightness (Cytometry A. 2004 Feb; 57 (2): 113-9 .). In addition, a method has also been considered in which fluorescent proteins are introduced into cells to establish cell division visualization cells and thereby evaluate the cell cycle (Japanese Patent Application Laid-Open No. 2004-187530). In addition, a method of visualizing intracellular proteins by immunostaining and analyzing their behavior is also generally performed. Immunostaining is a technique for detecting the localization of a target protein in cells and tissues using a specific binding reaction called an antigen-antibody reaction. There are two types of immunostaining methods: a direct method in which an enzyme or fluorescent substance is directly bound to a primary antibody that is a specific antibody against an antigen, and an indirect method in which a secondary antibody that is an antibody against the primary antibody is used for visualization. . The cell cycle consists of the following steps: synthesis preparation period (hereinafter referred to as G1 period) → synthesis period (hereinafter referred to as S period) → division preparation period (hereinafter referred to as G2 period) → division period (hereinafter referred to as M period). In the G1 phase, the amount of DNA is 2N (= 46 chromosomes), and the DNA is uniformly distributed in the nucleus. In the S phase, DNA is synthesized and changes from 2N to 4N. In the G2 phase, the amount of DNA becomes 4N and is uniformly distributed in the nucleus, and in the M phase, the DNA aggregates and becomes fluorescently stained, increasing its brightness. The above immunostaining method uses the amount and aggregation of DNA linked to the cell cycle, and the imaging cytometer creates a scattergram with the horizontal axis representing the DNA content and the vertical axis representing the maximum luminance. This is a method for judging the cell cycle based on the distribution. In this way, the cell cycle is identified and analyzed based on DNA content, and evaluation of new drugs for cancer and tumor cells and evaluation of malignancy, prognosis, malignancy progression by DNA aneuploidy analysis, drugs Is also important in determining which phase of the cell cycle works well.
JP2004-187530

However, when recognizing and analyzing a cell with an imaging cytometer, the outline of the cell is drawn based on the intensity of nuclear staining. An imaging cytometer collects data of thousands or tens of thousands of cells, but unlike a flow cytometer, individual cells are not necessarily isolated, and a plurality of cells may be in contact with each other. For this reason, in the conventional method, many cells are often recognized as one cell in a lump, and in such a case, it is difficult to separate and recognize each cell individually. Mass of cells, since the area than when it recognizes one cell is several times larger, but come out also exclude chunks from data while visually confirming which it takes time and effort, in a lump recognition The more cells that are being used, the more difficult it is to acquire accurate data.

  Therefore, the present invention has been made in view of such circumstances, and the object of the present invention is to extract useful data for cell cycle analysis without excluding it even if the cell is recognized as a mass. An object of the present invention is to provide a cell cycle analysis method capable of achieving this.

  To achieve the above objective, cell cycle-dependent biomolecules are stained and used as markers, and at the same time, the amount of nuclear DNA is measured to extract useful data from cells recognized as clumps. We investigated to determine the behavior and cell cycle of dependent biomolecules more clearly. As a result, the present inventors have found a method by which useful information can be obtained from a cell group that has been recognized as a lump by conventional cell cycle analysis and it has been difficult to obtain accurate data, and has led to the present invention.

That is, the cell cycle analysis method of the present invention uses a fluorescence to visualize cell nuclei in a cell population, and uses a fluorescence to express a biomolecule specifically expressed in a specific cell cycle in the cell in the cell population. Visualizing the cell, recognizing cells based on the fluorescence intensity of the cell nucleus, the total amount of fluorescence intensity of the cell nucleus in the cell population, and the total amount of fluorescence intensity of the biomolecule in the cell population at the same time Measuring the ratio of these cells and calculating the ratio thereof to identify the proportion of cells in the specific cell cycle among the cells in the cell population . Even when it was recognized as a mass cell, by performing the identification based on the total amount of fluorescence luminance, it was possible to grasp the total number of cells and the ratio of the total number of cells in a specific cell cycle. Characterized by the symptoms.

In a preferred embodiment of the cell cycle analysis method of the present invention, the image analysis of the cell population is performed with a microscope or an imaging cytometer.

  In a preferred embodiment of the cell cycle analysis method of the present invention, the specific cell cycle is M phase, and the biomolecule specifically expressed in the M phase is phosphorylated histone H3 (serine 10), phosphorus It is at least one selected from the group consisting of oxidized histone H3 (serine 28), phosphorylated CENP-A (serine 7), methylated histone H4 (lysine 20), cyclin B1, and aurora kinase. .

In a preferred embodiment of the cell cycle analysis method of the present invention, visualization of the biomolecule by fluorescence is performed by immunofluorescence staining.

In a preferred embodiment of the cell cycle analysis method of the present invention, visualization of the biomolecule by fluorescence is performed by introducing a fluorescent protein into the cell .

  According to the cell cycle analysis method of the present invention, there is an advantageous effect that useful data regarding the cell cycle can be acquired from cells recognized as a lump in an image. That is, according to the cell cycle analysis method of the present invention, it is possible to more accurately determine the behavior of cell cycle-dependent biomolecules, and it is advantageous that useful information can be extracted from cells recognized as a cluster. There is an effect.

  Furthermore, according to the cell cycle analysis method of the present invention, by identifying and analyzing the cell cycle, the evaluation of novel drugs and the evaluation of malignancy, prognosis, malignancy progression by DNA aneuploidy analysis, There is an advantageous effect that it is possible to determine which period of the cycle works well.

  The cell cycle analysis method of the present invention includes a step of visualizing a cell nucleus, a step of visualizing a biomolecule specifically expressed in a specific cell cycle, and numerical data of the cell nucleus obtained by visualizing the cell nucleus, The specific cell cycle ratio in the cell population is identified using the numerical data of the biomolecule obtained by visualizing the biomolecule.

  The method for visualizing cell nuclei is not particularly limited.

  The method for visualizing biomolecules that are specifically expressed in a specific cell cycle is not particularly limited. For example, a protein expressed in a cell cycle-dependent manner is immunostained with a fluorescent dye, or a fluorescent protein is expressed. The method of using the method of introduce | transducing into a cell etc. can be mentioned. In a preferred embodiment, the biomolecule is visualized by immunofluorescence staining.

  In the present invention, the percentage of cells in the specific cell cycle in the cell population, including massive cells, is identified using the numerical data of the cell nuclei thus obtained and the numerical data of the biomolecules. According to such a method, it is possible to successfully solve the problems of the conventional method in which a large number of cells are determined as one cell in a lump. Such data can be obtained by visualizing cell nuclei or biomolecules, e.g., by staining, and based on the staining fluorescence intensity (intensity), the total number of cells and the total number of cells in a specific cell cycle. It becomes possible to grasp the ratio.

  In order to explain the present invention in more detail, it will be described in comparison with a conventional method. FIG. 3 is a diagram showing a scattergram according to a conventional cell cycle analysis method. In FIG. 3, a threshold is set for the staining fluorescence intensity of cell nuclei with an imaging cytometer, and a scattergram when a cell is recognized by the set threshold (the horizontal axis is the total amount of fluorescence brightness (DNA content), the vertical axis Indicates the maximum fluorescence brightness (chromosome aggregation degree). The portion surrounded by a red line indicates cells with a DNA content of 4N, that is, G2 / M phase, but more than 4N, that is, many cells are detected on the right side. This is because a plurality of cells are recognized as one cell. In the conventional method, a DNA having a low degree of aggregation in the 4N portion, that is, one having a low maximum luminance is judged as the G2 phase, and a DNA aggregate is judged as the M phase.

  On the other hand, in the present invention, even when a plurality of cells as shown in FIG. As a process, cell nuclei and cell cycle-dependent biomolecular markers are visualized. Numerical data of cell nuclei obtained by visualization, for example, the total amount of nuclear staining and the total amount of cell cycle-dependent biomolecules are measured. For the measurement, a device capable of recognizing a cell image and measuring the amount of fluorescence, for example, a microscope, an imaging cytometer, or the like can be used.

It is possible to calculate the ratio by calculating the total amount of cell cycle-dependent biomolecules / total amount of nuclear staining from the last measured data. Thus, the visible cell cycle dependent biological molecules and DNA content was measured simultaneously, the cell cycle dependent biological molecule by a marker, cell cycle-dependent biomolecules in the entire cell population The behavior can be judged more accurately, and useful information can be extracted from the cells recognized as a lump.

  In a preferred embodiment, the specific cell cycle is M phase, and the biomolecule specifically expressed in the M phase is phosphorylated histone H3 (serine 10), phosphorylated histone H3 (serine 28), It is at least one selected from the group consisting of phosphorylated CENP-A (serine 7), methylated histone H4 (lysine 20), cyclin B1, and aurora kinase.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not intended to be interpreted as being limited to the following examples.

Example 1
Plate creation
HeLa cells (human cervical cancer cells) were cultured and trypsinized to prepare a cell suspension, which was then implanted into a 96-well multiplate at a concentration of 2 × 10 ⁴ / well. After about 4 hours, it was confirmed that the cells adhered, and the drug nocodazole that synchronizes the cell cycle in the M phase was serially diluted. After culturing at 37 ° C. in a 5% CO ₂ incubator for about 16 hours, the cells were fixed with paraformaldehyde having a final concentration of 2%.
Immunostaining for cell cycle specific markers
After blocking with 1% BSA / 0.1% Tween20 / PBS, the phosphorylated histone H3 (serine 10) (Upstate, catalog number 06-570), known as an M-phase specific marker, is diluted 200-fold to obtain a primary antibody. And reacted at 4 ° C. overnight. Thereafter, the plate was washed with PBS and reacted with Alexa488 goat anti-rabbit IgG (Molecular Probes, catalog number A11008) as a secondary antibody at room temperature for 1 hour.
Cell nucleus staining After staining of the secondary antibody, the cells were washed with PBS, and 5 μM DRAQ5 ^™ (Biostate, catalog number BOS-889-001-R200) was diluted 1000 times to stain the nucleus.
Data acquisition using an imaging cytometer Plates stained with cell cycle-specific markers and nuclei were acquired using an imaging cytometer (iCyte ^™ , OLYMPUS), and the target nuclei and phosphorylated histone H3 (serine 10) were detected. Fluorescence was confirmed. Thereafter, data on the total fluorescence amount of nuclear staining, the total fluorescence amount of phosphorylated histone H3 (serine 10), and the number of cells were obtained, and the results of calculating the ratio of phosphorylated histone H3 (serine 10) are shown in FIG. FIG. 2 is an average value of the results obtained when the same conditions were performed in three wells. The vertical axis represents the total amount of cell cycle-dependent biomolecules / total amount of nuclear staining, and the horizontal axis represents the concentration of the reagent nocodazole that synchronizes the cell cycle with the M phase. When measured using the method of the present invention, it can be seen that the higher the concentration of nocodazole, the higher the proportion of cells in the M phase. The purpose of this example is to quantify (graph) the increase in the M phase depending on the concentration of nocodazole. Nocodazole is a reagent that synchronizes cells to M phase by inhibiting microtubules during cell division, so increasing the amount of nocodazole added increases the number of cells in M phase.
In FIG. 2, as the concentration of nocodazole increases, in addition to the effect that nocodazole synchronizes the cells into the M phase, the number of cells in the M phase then decreases, which indicates that toxicity began to appear in the cells. Is considered to be the cause. That is, when toxicity appears in cells, it is considered that the cell cycle is stopped or apoptosis is caused.
FIG. 1 shows a partially enlarged view of an image acquired by an imaging cytometer. Red fluorescence indicates fluorescence of cell nucleus staining, and green tendency indicates fluorescence of phosphorylated histone H3 (serine 10). Here, the threshold is set according to the fluorescence intensity of nuclear staining, and the outline of the cell is automatically drawn, and it can be seen that there is a part that recognizes the cell as a lump. Among the cells recognized as lumps, there is phosphorylated histone H3 (serine 10) (cells emitting green fluorescence), but in actual analysis, the area of the cells is recognized to be extremely large. Therefore, it will be excluded from the data. In the present invention captures the fluorescence intensity of these cells as numerical data were calculated fluorescence amount / percentage of a fluorescent total nuclear staining histone H3 phosphorylation of phosphorylated histone H3 (Ser10) (Figure 2). As a result, the information of phosphorylated histone H3 could be extracted from the cells in a lump, and the fluctuation process of phosphorylated histone H3 in the cell cycle during nocodazole treatment could be shown more clearly.

  The present invention is important in evaluating new drugs for cancer and tumor cells, evaluating malignancy, prognosis and malignant progression by DNA aneuploidy analysis, and determining in which stage of the cell cycle the drug works well. It can provide information and can be applied in a wide range of fields.

FIG. 1 shows a partially enlarged view of an image acquired by an imaging cytometer. FIG. 2 shows the result of data acquisition by the image cytometer. FIG. 3 shows a scattergram according to a conventional cell cycle analysis method.

Claims (5)

Visualizing cell nuclei in a cell population using fluorescence;
Visualizing, using fluorescence, a biomolecule that is specifically expressed in a specific cell cycle within the cells of the cell population;
Recognizing cells based on the fluorescence intensity of the cell nuclei;
By simultaneously measuring the total amount of fluorescence intensity of the cell nuclei in the cell population and the total amount of fluorescence intensity of the biomolecules in the cell population and calculating the ratio thereof, the identification of the cells in the cell population The step of identifying the proportion of cells in the cell cycle, and when the plurality of cells are recognized as one lump cell in the step of recognizing the cells, the identification based on the total amount of fluorescence luminance is performed. A method for analyzing a cell cycle in vitro, characterized in that the total number of cells and the ratio of the total number of cells in a specific cell cycle are grasped.   The method according to claim 1, wherein image analysis of the cell population is performed with a microscope or an imaging cytometer.   The specific cell cycle is M phase, and biomolecules specifically expressed in the M phase are phosphorylated histone H3 (serine 10), phosphorylated histone H3 (serine 28), phosphorylated CENP-A (serine The method according to claim 1 or 2, which is at least one selected from the group consisting of 7), methylated histone H4 (lysine 20), cyclin B1, and aurora kinase.   The method according to any one of claims 1 to 3, wherein visualization of the biomolecule by fluorescence is performed by immunofluorescence staining. The method according to any one of claims 1 to 3, wherein visualization of the biomolecule by fluorescence is performed by introducing a fluorescent protein into a cell.